This invention relates to delivery of a bioactive agent. More particularly, the invention relates to a composition and method for delivering bioactive agents, such as DNA, RNA, oligonucleotides, proteins, peptides, and drugs, by facilitating their transmembrane transport or by enhancing their adhesion to biological surfaces. It relates particularly to a novel cationic lipopolymer comprising a branched polyethylenimine (PEI), a cholesterol derived lipid anchor, and a biodegradable linker which covalently links the branched PEI and cholesterol derived lipid anchor. One example of such a novel lipolymer is poly {(ethylene imine)-co-[N-2-aminoethyl)ethylene imine]-co-[N-(N-cholesteryloxycabonyl-(2-aminoethyl))ethylene imine]} (hereafter referred to as xe2x80x9cPEACExe2x80x9d). The cationic lipopolymers of the present invention can be used in drug delivery and are especially useful for delivery of a nucleic acid or any anionic bioactive agent.
Gene therapy is generally considered as a promising approach not only for the treatment of diseases with genetic defects but also in the development of strategies for treatment and prevention of chronic diseases such as cancer, cardiovascular disease and rheumatoid arthritis. However, nucleic acids as well as other polyanionic substances are rapidly degraded by nucleases and exhibit poor cellular uptake when delivered in aqueous solutions. Since early efforts to identify methods for delivery of nucleic acids in tissue culture cells in the mid 1950""s, steady progress has been made towards improving delivery of functional DNA, RNA, and antisense oligonucleotides in vitro and in vivo.
The gene carriers used so far include viral systems (retroviruses, adenoviruses, adeno-associated viruses, or herpes simplex viruses) or nonviral systems (liposomes, polymers, peptides, calcium phosphate precipitation and electroporation). Viral vectors have been shown to have high transfection efficiency when compared to non-viral vectors, but due to several drawbacks, such as targeting only dividing cells, random DNA insertion, their low capacity for carrying large sized therapeutic genes, risk of replication, and possible host immune reaction, their use in vivo is severely limited.
Compared to viral vectors, nonviral vectors are easy to make and less likely to produce immune reactions, and there is no replication reaction required. There has been increasing attention focused on the development of safe and efficient nonviral gene transfer vectors, which are either polycationic polymers or cationic lipids. Polycationic polymers such as poly-L-lysine, poly-L-omithine and polyethyleneimine (PEI), that interact with DNA to form polyionic complexes, have been introduced for use in gene delivery. Various cationic lipids have also been shown to form lipoplexes with DNA and induce efficient transfection of various eukaryotic cells. Among such kinds of synthetic vectors, cationic lipids are widely used because it is possible to design and synthesize numerous derivatives that are outstanding in the aspects of transfection efficiency, biodegradability and low toxicity. Many different cationic lipids are commercially available and several lipids have already been used in the clinical setting. Among them, cationic cholesterol derivatives are known to be very useful because of their high transfection efficiency in vitro. Although the mechanism of this transfection activity is not yet clear, it probably involves binding of the DNA/lipid complex with the cell surface via excess positive charges on the complex. Cell surface bound complexes are probably internalized and the DNA released into the cytoplasm of the cell from an endocytic compartment.
However, it is not feasible to directly extend in vitro transfection technology to in vivo applications. Relative to in vivo use, the biggest drawback of the diether lipids, such as N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl ammonium chloride (DOTMA) or Lipofectin is that they are not natural metabolites of the body, and are thus not biodegradable and they are toxic to cells. In addition, it has been reported that cationic lipid transfection is inhibited by factors present in serum and thus it is an ineffective means for the introduction of genetic material into cells in vivo.
An ideal transfection reagent should exhibit a high level of transfection activity without needing any mechanical or physical manipulation of the cells or tissues. The reagent should be non-toxic, or minimally toxic, at the effective dose. In order to avoid any long-term adverse side-effects on the treated cells, it should also be biodegradable. When gene carriers are used for delivery of nucleic acids in vivo, it is essential that the gene carriers themselves be nontoxic and that they degrade into non-toxic products. To minimize the toxicity of the intact gene carrier and its degradation products, the design of gene carriers needs to be based on naturally occurring metabolites. U.S. Pat. No. 5,283,185, to Epand et al. (hereafter the ""185 patent), discloses a method for facilitating the transfer of nucleic acids into cells comprising preparing a mixed lipid dispersion of a cationic lipid. 3xcex2[N-(Nxe2x80x2,Nxe2x80x3-dimethylaminoethane)-carbamoyl]cholesterol(DC-cholesterol) with a co-lipid in a suitable carrier solvent. The method disclosed in the ""185 patent involves using a halogenated solvent in preparing a liposome suspension. For pharmaceutical applications, residues of halogenated solvents cannot be practically removed from a preparation after having been introduced. U.S. Pat. No. 5,753,262, (hereafter the ""262 patent) discloses using the acid salt of the lipid 3xcex2[N-(Nxe2x80x2,Nxe2x80x3-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol) and a helper lipid such as dioleoyl phosphatidylethanolamine (DOPE) or dioleoylphosphatidylcholine (DOPC) to produce effective transfection in vitro. In addition, these cationic lipids have been proven less efficient in gene transfer in vivo.
Because of their sub-cellular size, nanoparticles are hypothesized to enhance interfacial cellular uptake, thus achieving in a true sense a xe2x80x9clocal pharmacological drug effect.xe2x80x9d It is also hypothesized that there would be enhanced cellular uptake of drugs contained in nanoparticles (due to endocytosis) compared to the corresponding free drugs. Nanoparticles have been investigated as drug carrier systems for tumor localization of therapeutic agents in cancer therapy, for intracellular targeting (antiviral or antibacterial agents), for targeting to the reticuloendothelial system (parasitic infections), as an immunological adjuvant (by oral and subcutaneous routes), for ocular delivery with sustained drug action, and for prolonged systemic drug therapy.
In view of the foregoing, it will be appreciated that providing a gene carrier that is non-toxic, biodegradable, and capable of forming nanoparticles, liposomes, or micelles for gene therapy and drug delivery, is desired. The novel gene carrier of the present invention comprises a novel cationic lipopolymer comprising a branched polyethylenimine(PEI), a cholesterol derived lipid anchor, and a biodegradable linker which covalently links the branched PEI and cholesterol derived lipid anchor. The lipolymer of the present invention is useful for preparing a cationic liposome, or a cationic micelle for drug delivery, especially for delivery of nucleic acids, other anionic bioactive molecules or both and is readily susceptible to metabolic degradation after incorporation into the cell.
The present invention provides a biodegradable cationic lipopolymer, having reduced in vivo and in vitro toxicity, for delivery of drugs or other bioactive agents to an individual in need thereof.
The present invention also provides a cationic lipopolymer for delivery of nucleic acids which carries out both stable and transient transfection of polynucleotides such as DNA and RNA into cells more effectively.
The biodegradable, non-toxic cationic lipopolymer of the present invention comprises a branched polyethylenimine (PEI), a cholesterol derived lipid anchor, and a biodegradable linker which covalently links the branched PEI and cholesterol derived lipid anchor. Preferably, the average molecular weight of the branched PEI is within a range of 600 to 25,000 Daltons. The branched PEI is preferably conjugated to the cholesterol derivative by an ester bond. The molar ratio of the branched PEI to the conjugated cholesterol derivative is preferably within a range of 1:1 to 1:20.
By adjusting the molecular weight of the branched PEI and the molar ratio of the branched PEI to conjugated cholesterol derivative, the resultant lipolymer can be either water soluble or water insoluble. For example, to obtain a water soluble lipopolymer, the average molecular weight of the branched PEI is preferably within a range of 1800 to 25,000, and the molar ratio of the branched PEI to the conjugated cholesterol derivative is preferably within a range of 1:1 to 1:5. To obtain a water insoluble lipopolymer, the average molecular weight of the branched PEI is preferably within a range of 600 to 1800, and the molar ratio of the branched PEI to the conjugated cholesterol derivative is preferably within a range of 1:1 to 1:2. Although a cholesterol derived lipid anchor is preferred in the present invention, other lipophilic moieties may also be used, such as C12 to C18 saturated or unsaturated fatty acids.
The biodegradable lipopolymers can be synthesized by relatively simple and inexpensive methods. These cationic lipopolymers invention can spontaneously form discrete nanometer-sized particles with a nucleic acid, which can promote more efficient gene transfection into mammalian cell lines than can be achieved conventionally with Lipofectin and polyethyleneimine. The lipopolymer the present invention is readily susceptible to metabolic degradation after incorporation into animal cells. Moreover, the water soluble cationic lipopolymer can form an aqueous micellar solution which is particularly useful for systemic delivery of various bioactive agents such as DNA, proteins, hydrophobic or hydrophilic drugs. The water insoluble lipopolymer can form cationic liposomes with a helper, which is particularly useful for local drug delivery. Therefore, the biocompatible and biodegradable cationic lipopolymer of this invention provides an improved gene carrier for use as a general reagent for transfection of mammalian cells, and for the in vivo application of gene therapy.
The present invention further provides transfection formulations, comprising a novel cationic lipopolymer, complexed with a selected nucleic acid in the proper charge ratio(positve charge of the lipopolymer/negative charge of the nucleic acid) that is optimally effective for both in vivo and in vitro transfection. Particularly, for systemic delivery, the charge ratio (+/xe2x88x92) is preferably 5/1 to 1/1; for local delivery, the charge ratio (+/xe2x88x92) is preferably 3/1 to 0.5/1.
This invention also provides for a method of transfecting, both in vivo and in vitro, a nucleic acid into a mammalian cell. The method comprises contacting the cell with a cationic lipopolymer or liposome:nucleic acid complexes as described above. In one embodiment, the method uses systemic administration of the cationic lipopolymer or liposome:nucleic acid complexes into a warm blooded animal. In a preferred embodiment, the method of transfecting uses intravenous administration of the cationic lipopolymer or liposome:nucleic acid complexes into a warm blooded animal. In a particularly preferred embodiment, the method comprises intravenous injection of water soluble PEACE/pDNA and PEACE:DOPE liposomes/pDNA complexes into a warm blooded animal.